Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains ix, 87 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 75-87).

This study investigated the biological activity of a macrophage activating factor (MAF) produced by activated lymphocytes from the rainbow trout (Oncorhynchus mykiss) and attempts to discover its molecular source. Peripheral blood lymphocytes were shown to release factors with MAF activity following incubation with a variety of stimulants and were subsequently shown to activate macrophages using at least two different methods, the nitroblue tetrazolium (NBT) colourimetric assay and the luminol-dependent chemiluminescent assay. The latter technique detected an immediate response which decayed over a 40 minute period on the addition of cell-free supernatants from activated lymphocytes to macrophages. A number of molecular approaches, including degenerate PCR primer amplification, DNA cross-hybridisation and cDNA library screening were used in this study to try to isolate any cytokine genes from Oncorhynchus mykiss. As a control β-actin cDNA was successfully amplified from Oncorhynchus mykiss using primers based on the salmon sequence. The Oncorhynchus mykiss orthologue of IFN-y was initially targeted. However, although a PCR product of the appropriate size was amplified using degenerate primers based on mammalian and avian IFN-y sequences, the sequence was not related to IFN-y or any other known Oncorhynchus mykiss sequence. A similar strategy was used to try and amplify the Oncorhynchus mykiss orthologue of mammalian IL-15. Again despite amplification of a DNA fragment of approximately the correct size there appeared to be no relationship between it and the known IL-15 sequences. As an alternative strategy a cDNA library from stimulated peripheral blood lymphocytes (PBLs) was constructed and screened using cDNA probes derived from stimulated and non-stimulated PBLs in order to detect mRNAs which might have been upregulated as a result of in vitro stimulation. A number of positive clones were obtained from the differential screening of the library including cDNAs showing similarity to other unidentified fish sequences as well as to a number of proteins predicted to be involved in regulation of cell proliferation, neocorticogenesis and embryo development. Additionally. the library was also screened using ovine cytokine cDNA probes. although no positively hybridising clones were obtained. The ovine IFN-y gene was also used to probe genomic DNA from Oncorhynchus mykiss. but unlike previous studies with human IFN-y gene no hybridisation between the ovine IFN-y gene and Oncorhynchus mykiss DNA was observed. This investigation highlights the potential difficulties of using various molecular strategies such as DNA cross-hybridisation or PCR techniques for the cloning of fish cytokine sequences. Consequently, future strategies for cloning fish cytokine genes may require targeting the biological activity through expression libraries.

M.Sc. (Zoology) / Please refer to full text to view abstract

The marked differences in response to water current, exhibited by juvenile rainbow trout migrating into Loon Lake from its outlet and inlet streams, were studied both in the field and in experimental laboratory apparatus. All available evidence argued against genetically discrete outlet and inlet stocks, each maintaining different innate responses to water current. Difference in water temperature between streams was shown, in field and laboratory experiments, to regulate direction of juvenile trout migration through action on behaviour associated with downstream movement, maintenance of position and upstream movement. In laboratory experiments with cool (5°, 10° C.) flowing water, recently emerged fry rarely made contact with the stream bottom in darkness and exhibited much more downstream movement than in warm (> 14°C.) water. In cool streams of the Loon Lake system (daily mean consistently < 13°C.) large numbers of recently emerged fry moved downstream in darkness. Laboratory experiments indicated that combination of cool water (10°C.) and long day length (sixteen hours) induced downstream movement of fingerlings. In the field, fingerlings moved downstream largely in late spring and summer in cool streams of the Loon Lake system. In laboratory experiments with warm (15°, 20°C.) flowing water, recently emerged fry made frequent contact with the stream bottom in darkness and exhibited much less downstream movement than in cool (10°C.) water. In the warm outlet stream (daily mean in summer usually > 15°C.) recently emerged fry maintained position in darkness. Laboratory experiments suggested that short day length (eight hours) may facilitate maintenance of position exhibited by fingerlings in streams during late autumn and winter. Upstream movement of fry tested in the field and laboratory was most pronounced in warm water (>14°C). Fingerlings subjected to rapid 5°C. increases in water temperature in an experimental stream exhibited an immediate increase in upstream movement. Upstream movement in summer of large fry and fingerlings occurred only in the warm outlet stream; daily periodicity of upstream movement was positively correlated with sharp rises in water temperature. Evidence examined from four other widely separated stream systems indicated an environmental control of migration in juvenile rainbow trout similar to that demonstrated in the Loon Lake stream system. Possible mechanisms and interaction of factors controlling migratory patterns between and within streams are discussed. Significance of the predominant role played by temperature is considered. / Science, Faculty of / Zoology, Department of / Graduate

Criteria for interpretation of scales of steelhead (Salmo gairdneri gairdneri) and for their use in back calculation of early life history were established. With these criteria, features of the life history of 784 steelhead from the anglers' catches of the Chilliwack River, B. C. were studied. Age composition of adult steelhead runs from 1948 to 1953 was uniform except for the suggestion of a slightly dominant year class, represented as 4 year old fish in 1950, 5 year old fish in 1951 and 6 year old fish in 1952. There was no suggestion that fish of any particular age, life history, sex or length tend to return as adults at any particular season of the year. The age and size of smolts at migration is variable, with the majority however in the 2 and 3 year age groups and ranging in size from 15 to 20 cms. Smolt migration usually occurs in March, with a small proportion of late migrants in August. Spawning frequency of adults was reviewed. Females generally spawn at an earlier age and more frequently than males. It is concluded that the variability in the life history of the steelhead acts as a safeguard in its conservation. / Science, Faculty of / Zoology, Department of / Graduate

At a late stage of spermatogenesis a sperm-specific protein, protamine, is synthesized in the testis of salmonid fish and progressively replaces histones in combination with DNA. Protamine has a molecular weight near 5,000 and contains 2/3 of its total amino acid residues as arginine. Studies on the biosynthesis of protamine have been made on the rainbow trout (Salmo gairdnerii) testis. Successive column chromatography on Bio-Gel P-10 and CM- cellulose has been employed to isolate and characterize newly synthesized labelled protamine. Newly synthesized protamine is phosphorylated and is eluted earlier from the CM-cellulose column than mature protamine. However, the two forms of protamine chromatograph coin-cidentally when newly synthesized protamine is first treated with alkaline phosphatase. Protamine is separated into three components on CM-cellulose and the amino acid compositions of the components are very similar. The relative amounts of the components present in the testis nuclei are different at different stages of spermatogenesis and the synthesis of each component appears to be independently controlled. This suggests that the components, while chemically very similar, are the products of separate structural genes and may have different functions. By pulse labelling testis cell suspensions for different lengths of time and analyzing the amount of ¹⁴C- protamine found in the cytoplasm and in the nucleus, the site of protamine synthesis can be shown to be in the cytoplasm. Further, a cell-free, isolated post-mitochondrial cytoplasmic fraction can incorporate ¹⁴C-arginine into whole protamine molecules, while both an isolated nuclear fraction and high-speed supernatant were relatively inactive. This indicates that protamine synthesis occurs on cytoplasmic microsomes. Sedimentation analysis of pulse-labelled testis ribososes indicates that protamine is synthesized on a class of small polysomes, the disomes, sedimenting at 120S. While dimeric ribosomes investigated in various tissues have been shown to be inactive artefacts formed during isolation, the disomes in trout testis have been demonstrated to be a functional class of polysomes. They are not dissociable at 1 mM Mg⁺⁺ ion concentration, are not the breakdown product of larger polysomes, nor are they produced by interaction with free protamine. These disomes contain the major quantity of nascent protamine and increase in number in the testis cells during the active protamine synthesizing stage of development. The probable function of protamine is for the packaging of DNA into the sperm head. The phosphorylation of protamine and the protamine components may serve to regulate this packaging process. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The Rainbow Range is a Late Miocene shield volcano (30 km diameter, 370 km³) whose stratiform flanks surround a complex central vent zone. Over a time span of 1-2 m.y., extrusion of highly fluid comendites and comenditic trachytes, along with minor mugearites and hawaiites, built up the gently sloping flanks. The viscosity of the peralkaline lavas was so low that their eruption produced a shield volcano rather than a composite cone. Comenditic trachytes (65.5 percent S10₂) are the lowest flows exposed on the north flank of the Rainbow Range. Chemical traits include high Na20 + K/jO (11 percent), moderately high AI₂O₃ (15 percent), low total iron as Fe₂0₃ (5 percent), and high Ba (300-1000 ppm). Thin flows of mugearite (54.9 percent Si0₂) rest on the comenditic trachytes. Comendites (68.7 percent Si02) uncon-formably overlie the mugearites and account for at least 75 percent of the volume of flows within the flank zone. These lavas are distinguished by lower AI2O3 (13 percent), higher total iron as Fe203 (7 percent), and extremely depleted Sr (1-10 ppm) and Ba (10-100 ppm). The termination of flank volcanic activity is recorded by the eruption of capping flows and related feeder dikes of hawaiite (50.1 percent Si0₂). Comenditic trachytes contain phenocrysts of anorthoclase (Or₂₅_₂₇), heden-bergite, and iron-titanium oxides in a groundmass of alkali feldspar, quartz, acmite, iron-titanium oxides, aenigmatite, and arfvedsonite. Comendites bear the phenocryst assemblage sanidine (Or₃₄_₃₇) + hedenbergite + fayalite + arfvedsonite set in a pilotaxitic groundmass of alkali feldspar, quartz, acmite, iron-titanium oxides, aenigmatite, and arfvedsonite. Continuous variation in major and trace element trends and feldspar compositions suggests that the hawaiite-mugearite-comenditic trachyte-comendite suite was derived from an alkali basalt parent, tapped several times as it underwent prolonged fractional crystallization in an intracrustal magma chamber. A best-fit mathematical model for the origin of the suite involves step-wise derivation of the lavas.in the order hawaiite > mugearite ? comenditic trachyte > comendite, with the main phases precipitating out in the order olivine, clinopyroxene, plagioclase, iron-titanium oxide, and alkali feldspar. Strontium isotopic evidence indicates that the peralkaline lavas were erupted soon after differentiation. The Rainbow Range and other peralkaline and alkaline volcanic centers of the Anahim volcanic belt are coeval.with calc-alkaline volcanic centers of the Pemberton volcanic belt. Together these belts outline the orientation and extent of the subducted Juan de Fuca plate during Late Miocene time. Volcanic activity in the Anahim belt may be related to a) an "edge effect" of the subducted Juan de Fuca plate, b) movement of the North American plate over a mantle hot spot at a rate of 2-3 cm/year, or c) an east-west trending rift zone. / Science, Faculty of / Earth, Ocean and Atmospheric Sciences, Department of / Graduate

Acute ammonia toxicity in rainbow trout was studied. This was carried out by injecting fish with various concentrations of ammonia dissolved in isotonic saline. In order to approximate conditions of natural toxicity, where ammonia would enter the fish at the gills, without the additional problems of environmental factors, the ammonium solutions were injected via a cannula implanted in the dorsal aorta. To determine if a differential toxicity existed in fish in relation to high pH and low pH ammonium solutions, ammonium bicarbonate and ammonium chloride were chosen for injection. Hydrogen ion, and total ammonia, concentrations were measured in blood sampled from the dorsal aorta, both before and after injection. In order to determine if, during the course of the injection, normal excretion rates would remove all of the injected ammonia, total ammonia and hydrogen ion concentrations were measured in blood sampled from both the dorsal and ventral aortae, and rates of extraction of ammonia from blood at the gills were calculated. Ammonia is toxic to fish. There was no significant difference between the dose of NH₄Cl and NH₄HCO₃ which killed fish. Therefore, unlike mammals, fish exhibited no differential toxicity to the ammonium compounds tested. Injection of NH₄Cl decreased pHa and injection of NH₄HCO₃ increased pHa. Both compounds increased the total ammonia concentration in the blood. Although in water the fraction of ammonia which exerts the toxic effects is unionized ammonia, within the fish it is the ionized fraction which exerts the toxic effects. The same dose of ammonium killed fish, but NH₄HCO₃-injected fish which survived had a much higher concentration of unionized ammonia in the blood than NH₄Cl-injected fish which died. Ammonia extracted from the blood in control fish was about one-fifth the amount which killed fish. This, together with the measured increases in blood ammonia following injection, demonstrate that, although ammonia is a normal excretory product of rainbow trout, the trout cannot increase excretion rates sufficiently to rid themselves of an ammonia load. Symptoms observed in fish following injection of ammonium solutions led to the conclusion that ammonia acts at the neural level. / Science, Faculty of / Zoology, Department of / Graduate

In this work, rainbow trout were used as a model system to examine the regulation of insulin (INS)- and insulin receptor (IR)-encoding mRNA expression profiles. INS- and IR-encoding mRNAs were isolated, cloned, and sequenced; and shown to be differentially expressed within and among multiple tissue types. Regulation was examined through various nutritional and hormonal treatments (in vivo and in vitro). A real-time quantitative-PCR assay was developed to measure the respective levels of mRNA expression. Fasting, growth hormone (GH), and somatostatin (SS) differentially regulated INS and IR mRNAs within selected tissues, in vivo. Glucose, GH, SS, and insulin-like growth factor-1 (IGF-I) differentially regulated INS and IR mRNAs within selected tissues, in vitro. The results of this dissertation research display the identification and differential regulation of multiple INS- and IR-encoding mRNAs and suggest that independent mechanisms may serve to regulate the various isoforms in a tissue-specific manner. Future studies are also suggested.

Metabolic fuel use in rainbow trout (𝘖𝘯𝘤𝘰𝘳𝘩𝘺𝘯𝘤𝘩𝘶𝘴 𝘮𝘺𝘬𝘪𝘴𝘴 W.) was investigated using closed system respirometry and proximate body analysis. During short term starvation (15 days, routine activity) the utilization of protein as a substrate, as determined by respirometry, increased from 14 to 24% of total fuel supply. However, even by the end of the experiment, the contribution of protein (24%) did not approach the classically reported values for fish of between 40 and 90%. Indeed, from respirometry data, during the first quarter of the experiment lipid contributed the majority of the fuel ( >60%) while carbohydrate contributed about 20%. Thereafter, lipid and carbohydrate became essentially equivalent in importance (about 37% each). However, from proximate body analysis, a more traditional fuel mixture was found (protein, 58%; lipid, 40%; carbohydrate, 2%) suggesting the possibility that the two procedures were measuring fundamentally different parameters. Instantaneous fuel use during sustainable swimming at different speeds was investigated by respirometry using three day test periods. While protein catabolism remained constant over time, and uniform between groups, its relative contribution tended to increase with time as total M₀₂ declined with sustained swimming. Protein catabolism was highest in nonswimming fish (30-45%) and lowest in the high speed swimmers (20%); lipid was the most abundant (41-55%) fuel used in all groups at all times. In the nonswimmers and lowspeed swimmers, lipid use tended to increase slightly over time whereas in the high speed swimmers, lipid use dropped from 54 to 44%. Carbohydrate use (up to 38%) was higher than predicted by earlier literature, but decreased greatly in both the nonswimmers and low speed swimmers over the three days, whereas in the high speed swimmers the contribution increased with time. The low speed swimmers from the last set of experiments were used as controls for the final set of experiments in which another group of fish were trained for two weeks at 1.0 L·s⁻¹ prior to testing using an otherwise similar regime. Even though there was no difference in gas exchange, the make-up of the fuel mixture was different for the two groups. Protein use was significantly lower, while lipid use was higher in the trained fish. In addition, relative protein use in the trained fish was constant over the three day period, a feature found only in the the high speed swimmers of the previous experiment. A critical evaluation of the respiratory quotient is given since its use by fish physiologists has been without complete conversion from that used by the mammal physiologists. In addition, the often quoted term "fuel use" is differentiated into 𝘪𝘯𝘴𝘵𝘢𝘯𝘵𝘢𝘯𝘦𝘰𝘶𝘴 𝘧𝘶𝘦𝘭 𝘶𝘴𝘦 and 𝘤𝘰𝘮𝘱𝘰𝘴𝘪𝘵𝘪𝘰𝘯𝘢𝘭 𝘧𝘶𝘦𝘭 𝘶𝘴𝘦 since the two describe fundamentally different principles, though this has not always been recognized in the literature. / Thesis / Master of Science (MS)